表达绿色荧光蛋白基因的花鲈胚胎干细胞株的建立及其体外分化

以带有绿色荧光标记的基因(pCMV-EGFP)为报告基因,用Genejammer、Genejuice和Metafectene三种脂质体介导花鲈胚胎干细胞(LJES1)的基因转移.实验发现,Genejammer介导的细胞转化效率最高,高达27.3%,其余分别为12.1%和5.3%.转移绿色荧光蛋白(GFP)基因的LJES1细胞经过药物筛选和单克隆化培养,获得了表达GFP基因的阳性克隆细胞株,经PCR对GFP阳性细胞株的基因组DNA及提取的RNA扩增,获得了目的条带,证实了GFP基因已经整合到LJES1细胞的基因组中,并获得了正常的表达.通过体外诱导,GFP阳性细胞能够分化为神经细胞、肌肉细胞、成纤维细胞等,用悬滴法培养获得了GFP阳性细胞的拟胚体,证实了经过长期的药物筛选后,LJES1细胞仍然保持着发育的多能性.这一研究,为进一步利用海水鱼类胚胎干细胞进行遗传操作及基因工程的研究提供了方法上的探索.

Establishment and differentiation in vitro of sea perch embryonic stem cell line expressing GFP gene

This paper reports the mediated transformation efficiency for pCMV-EGFP gene transferred into embryonic stem (ES) cells derived from sea perch (LJES1) by three lipsomes of Genejammer, Genejuice and Metafectene. It indicates that the efficiency of Genejammer-mediated transformation of LJES1 was the most high. It fits for gene transfer of LJES1. The highest efficiency values of Genejammer, Genejuice, Metafectene -mediated transformation of LJES1 are 27.3%, 12.1%, and 5.3%, respectively. By drug selection, the embryonic stem cell line expressing GFP (GFP~+ LJES1) gene was obtained. By PCR technology, it was verified that GFP gene integrated into genomic DNA of LJES1, and expressed normally. Induced in vitro, GFP~+ LJES1 cells were differentiated into muscle cells, fibroblast cells, neuron cells, etc. Cultured in vitro, GFP~+ LJES1 cells came into embryoid bodies, it was verified that LJES1 cells still retained pluripotenty through drug selection for long-term. By the research, the ES cell methods and technology of genetic modifications in vitro through extrinsic gene integration were developed.